The research goal is to develop ion mobility-mass spectrometry (IM-MS) for high-throughput, proteomic scale applications. This proposal provides plans for developing IM-MS methods with enhanced throughput, sensitivity, resolution and information content to address analytical challenges of proteomics. The research is divided into sections that address the design of new instrumentation, incorporation of high-repetition rate (kHz) lasers to increase the IM-MS duty cycle to approximately 100%, which will increase sample throughput and reduce analysis time, and development of tandem MS techniques for simultaneous acquisition of peptide mass maps and peptide sequencing for rapid protein identification. The proposal also describes research collaborations with chemical-biology research groups that have immediate needs for proteomics-mass spectrometry. Such collaborations provide critical evaluations for the technique developmental research. These collaborations include drug design studies and structure determinations of key proteins involved in protein-drug interactions, viral protein-host protein interactions as well as membrane protein trafficking to the inner nuclear membrane, studies of how cells function by identifying expressed proteins and proteins components of multi-subunit complexes in E. coil, understanding bacterial pathogenesis by determining the proteins present on the surface of the bacteria, identifying the protein composition of seminal plasma fluids of fertile and sub-fertile stallions as well as degrees of post-translational modifications and key interacting protein and protein complexes, de novo sequencing of novel neuropeptides isolated from single neurons insects and other organisms, identifying functional state of ciracadian clock proteins and large hetero-multimeric protein complex call the "clockosome."